The central paradox of cardiac excitation-contraction (E-C) coupling is that Ca2+-induced Ca2+ release (CICR), an inherently self- regenerating process, is finely graded by surface membrane Ca2+ currents. Recently, the P.I. showed that the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors, RyRs) from cardiac muscle exhibit a unique adaptive behavior, characterized by the ability of individual channels/RyRs to respond to incremental increases in Ca2+ by transient bursts of activity. This phenomenon, termed RyR adaptation, could account for the graded nature of CICR in vivo and may be a fundamental property of all intracellular Ca2+ release channels, including the inositol trisphosphate receptors (IP3R). The goal of the proposal is to establish an independent research program that will provide insights into the mechanisms and physiological role of RyR adaptation. To carry out this project, the P.I. will take advantage of a unique combination of methodological tools, including recordings from single SR Ca2+ release channels in lipid bilayers and Ca2+ measurements in intact cells. Measurements of single channel activity in response to step [Ca2+] changes, induced by photolysis of caged-Ca2+, will be performed to define the dynamic properties of the RyR/Ca2+ channels (e.g. rate of adaptation, nonstationary Ca2+-dependence) in the presence of physiologically relevant ligands (e.g. ATP, Mg2+, Ca2+). Defining these "elementary" properties of Ca2+ release is essential for discriminating between alternative hypotheses of how CICR is regulated in vivo. For example, an adaptation with sufficiently rapid kinetics could operate by countering the intrinsic positive feedback of CICR. A slow shift in RyR Ca2+ sensitivity could provide a basic for a different mechanism, which operates by fine-tuning the Ca2+ sensitivity of the release to maintain a stable graded CICR. To determine how single channel adaptation is expressed in situ, experiments involving measurements of global and local [Ca2_] will be performed in intact cardiomyocytes using patch- clamping combined with microfluorometry or confocal Ca2+ imaging. The molecular basis of adaptation will be determined by ascertaining whether adaptation is an inherent property of the RyR or a closely associated regulatory protein is involved in this phenomenon. A comprehensive mathematical model of RyR adaptation will be developed to help to understand the mechanism of this phenomenon. Understanding how Ca2+ release is regulated is important since it represents a strategic site for therapeutic intervention. Further, defining the mechanism of regulation of cardiac Ca2+ release channels has broader significance since the regulation of analogous channels (IP3Rs and RyRs) in other tissues are not well understood.